Understanding the mechanisms by which mucosal pathogens such as Vibrio cholerae colonize the human intestinal mucosa is key to the rational development of live-attenuated vaccine derivatives capable of inducing protective immunity against cholera and other enteric diarrheal diseases. Current parenteral vaccination strategies for these infections are largely ineffective. Although many V. cholerae genes required for intestinal colonization have been identified, the molecular mechanisms by which the proteins they encode promote vibrio adherence to host tissue are poorly understood. The studies described in this research proposal represent an approach to understand at the molecular level, the contribution of the V. cholerae TcpI "chemotaxis related" protein to the intestinal colonization properties of this important human pathogen. TcpI is a 75-kDa inner membrane protein that belongs to a large family of signal transducing proteins involved in bacterial chemotaxis. V. cholerae tcpI mutants display an altered pattern of toxin- coregulated pilus (TCP) and cholera toxin expression. Specifically, tcpI mutants produce the TCP colonization pilus and cholera toxin under environmental conditions normally associated with a lack of pilus/toxin production (media pH of 7.0-8.4). These data show that TcpI is a repressor of pilus/toxin production in Luria Broth at neutral to basic pH. This proposal outlines a series of experiments aimed at understanding the contributions of TcpI to vibrio pilus/toxin production. V. cholerae TCP and cholera toxin are critical virulence determinants. Since TcpI contributes to the production of these essential virulence factors, it is important to understand the structure and function of TcpI. Our long-term goal is to use the information regarding the properties of this protein in the development of improved methods for treating and preventing cholera enteric infections. There are two SPECIFIC AIMS in this proposal: (1) Define the features of TcpI that promote the synthesis of toxin/pilus using ELISA/vibrio agglutination assays;(2) Determine at what level TcpI acts within the ToxR/TcpP/ToxT regulatory cascade to influence virulence gene expression by over-expressing relevant regulators of toxin/pilus synthesis. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is a major cause of morbidity and disease worldwide. This bacterium has a complex regulatory cascade that temporally and spatially controls virulence factor synthesis in response to intraintestinal signals. The proposed studies will determine the mechanism by which a V. cholerae chemotaxis protein regulates cholera toxin and toxin co-regulated pilus synthesis in response to pH. The may allow the development of strategies to control the spread of this dread human disease.